Subcellular Tracking of Cathepsin D and Mitophagy

After CTL treatment for 24 h, breast cancer cells were fixed with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 for 7 min. After PBS washing, cells were blocked with PBS-T containing 10% FBS and 1% BSA for 1 h. To measure the release of cathepsin D into the cytosol, cells were incubated with anti-cathepsin D primary antibody (sc-6486, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight and followed by Alexa Flour 488-conjugated anti-goat IgG for 1 h (1:250, Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 1 μg/mL DAPI (Sigma, St. Louis, MO, USA) in 2% BSA for 1 min. To analyze the mitophagy induction through the interaction of mitochondria with the lysosome, cells were incubated with primary antibodies (anti-COX Ⅳ, #4850, 1:250, Cell Signaling, Danvers, MA, USA and anti-LAMP2, sc-18822, 1:250, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were incubated with Alexa Flour 488-conjugated anti-rabbit IgG and 594-conjugated anti-mouse IgG (1:250, Invitrogen, Carlsbad, CA, USA) for 1 h. The autophagosome-mediated mitophagy was analyzed by staining with anti-COX Ⅳ in GFP-LC3-transfected cells, followed by incubating with Alexa Flour 594-conjugated anti-rabbit IgG. To assess the integrity of lysosome, cells were treated with CTL for 24 h and stained with 50 nM Lysotracker Red DND-99 at 37 °C for 30 min. Fluorescence images were acquired using confocal microscopy (Carl Zeiss, Jena, Germany).